Affinity purification coupled with mass spectrometry proteomics (human)

Parental and ITCH KO HeLa cells were cultured in lysine- and arginine-free DMEM supplemented with dialyzed FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin/streptomycin and light (K0) lysine (38 μg/mL) and arginine (66 μg/ml). Heavy medium was the same except the light lysine was replaced with K8-lysine (L-Lysine, 2HCl U-13C U-15N, Cambridge Isotope Laboratories Inc). All cells were treated for 1 h with 1mM LLOMe (Sigma). Subsequently, cells were processed as described before (Fiskin et al., 2016). Briefly, cells were washed twice with ice-cold PBS and lysed in 5 ml denaturing lysis buffer (8 M Urea, 50 mM Tris pH 8, 50 mM NaCl, 1X PIC (protease inhibitor cocktail, EDTA-free, Roche), 50 µM DUB inhibitor PR-619 (Millipore)). Samples were incubated on ice for 10 min and then sonicated with 3x 20 s pulses. Following removal of non-solubilized material (15,000xg/10 min), differentially labeled lysates were mixed at equal ratios based on total protein determined by BCA (Pierce-Thermo; typically 25-35 mg of total protein). Following reduction with 5 mM DTT and alkylation with 10 mM chloroacetamide, lysates were digested with 5 ng/μl lys-C (Wako) for 1 h at room temperature. Subsequent digestion of peptides with trypsin (Promega) was performed as described (Villen and Gygi, 2008). Lyophilized peptides were resuspended in 1.5 ml IAP buffer (50 mM MOPS pH 7.4, 10 mM Na2HPO4, 50 mM NaCl) and centrifuged to remove any insoluble material (2500xg/5 min). The supernatant was incubated with anti-diGly antibody (32 μg/IP) conjugated to protein A agarose beads (Cell Signaling) for 1 h at 4°C. Unbound peptides were removed through 3x washing with IAP buffer and once with PBS. Bound material was eluted 4x with 50 µl 0.15% TFA and peptides were desalted using C18 stage-tip method (Rappsilber et al., 2003). Each sample was immunoprecipitated sequentially three times and each IP was analyzed separately by mass spectrometry. Peptides samples were separated on a nanoflow HPLC system (Thermo Scientific) using a 226 min gradient of 5-33% acetonitrile containing 0.5% acetic acid on custom filled C18 reversed-phase columns and analyzed on a hybrid ion-trap Orbitrap mass spectrometer (Orbitrap Elite, Thermo Scientific) using data-dependent acquisition selecting the most intense peaks from each full MS scan acquired in the Orbitrap for subsequent MS/MS while excluding peptides with unassigned charge states or charge states below +3 from fragmentation.